Polypeptide Variants

Abstract

The present invention relates to methods for selecting, obtaining or producing Fc variant polypeptides which show altered recognition for an Fc ligand (e.g., FcγR, CIq). Additionally, the Fc variant polypeptides may have altered antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) activity. The invention further provides methods and protocols for the application of said Fc variant polypeptides particularly for therapeutic purposes.

1 . A method of providing an Fc polypeptide variant with altered recognition of an Fc ligand and/or improved effector function compared with a parent Fc polypeptide, the method comprising:
 (a) providing mRNA molecules, each mRNA molecule comprising a nucleotide sequence encoding an Fc polypeptide variant and lacking an in-frame stop codon;   (b) incubating the mRNA molecules under conditions for ribosome translation of the mRNA molecules to produce encoded Fc polypeptide variants, whereby complexes each comprising at least mRNA and encoded Fc polypeptide variant are formed;   (c) bringing the complexes into contact with an Fc ligand that binds the parent Fc polypeptide, and selecting one or more complexes each displaying an Fc polypeptide variant able to bind the Fc ligand under the conditions of the selection;   (d) determining recognition or effector function of selected Fc polypeptide variant or variants, whereby one or more Fc polypeptide variants with improved Fc ligand recognition and/or improved effector function compared with the parent Fc polypeptide are obtained;   (e) retrieving mRNA from a selected complex; and   (f) amplifying and copying the retrieved mRNA into DNA encoding the selected Fc polypeptide variant.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the Fc ligand is an Fc receptor or C1q. 
     
     
         5 . The method of  claim 4 , wherein the Fc receptor is selected from FcγRI, FcγRII or FcγRIII families. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the altered recognition of an Fc ligand is reduced binding to FcγRIIb. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the altered recognition of an Fc ligand is improved binding to FcγRIIIa. 
     
     
         12 . The method of  claim 1 , wherein the effector function is ADCC or CDC. 
     
     
         13 - 16 . (canceled) 
     
     
         17 . The method of  claim 1 , wherein the Fc polypeptide variant comprises a variant human IgG Fc region. 
     
     
         18 . The method of  claim 17 , wherein the IgG Fc region is selected from IgG1, IgG2, IgG3 or IgG4. 
     
     
         19 - 22 . (canceled) 
     
     
         23 . The method of  claim 1  wherein the DNA encoding the selected Fc polypeptide variant. is provided in an expression system. 
     
     
         24 - 34 . (canceled) 
     
     
         35 . An Fc polypeptide variant having a mutation at two or more positions in the amino acid sequence of human wild-type sequence of SEQ ID NO: 1, wherein the residue provided at any one of said positions is selected from the following: 224N/Y, 225A, 228L, 230S, 239P, 240A, 241L, 243S/L/G/H/I, 244L, 246E, 247L/A, 252T, 254T/P, 258K, 261Y, 265V, 266A, 267G/N, 268N, 269K/G, 273A, 276D, 278H, 279M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I/F, 311R, 312N, 315D/K/S, 320R, 322E, 323A, 324T, 325S, 326E/R, 332T, 333D/G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S, 352A, 354A, 355W, 356G, 358T, 361D/Y, 362L, 364C, 365Q/P, 370R, 372L, 377V, 378T, 383N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F/P, 428T, 430K, 431S, 432P, 433P, 438L, 439E/R, 440G, 441F, 442T, 445R, 446A, 447E, wherein the variant has altered recognition of an Fc ligand and/or altered effector function compared with a parent Fc polypeptide, and wherein the numbering of the residues is that of the EU index as in Kabat. 
     
     
         36 . (canceled) 
     
     
         37 . The Fc polypeptide variant of  claim 35  comprising a set of mutations in the human wild-type sequence of SEQ ID NO: 1 selected from the group consisting of the following sets of mutations:
 (2) N276D, R292Q, V305A, I377V, T394A, V412A and K439E;   (3) P244L, K246E, D399G and K409R;   (4) S304G, K320R, S324T, K326E and M358T;   (5) F243S, P247L, D265V, V266A, S383N and T411I;   (6) H224N, F243L, T393A and H433P;   (7) V240A, S267G, G341E and E356G;   (8) M252T, P291L, P352A, R355W, N390D, S408G, S426F and A431S;   (9) P228L, T289A, L365Q, N389S and 5440G;   (10) F241L, V273A, K340Q and L441F;   (11) F241L, T299A, I332T and M428T;   (12) E269K, Y300H, Q342R, V422I and G446A;   (13) T225A, R301c, S304G, D312N, N315D, L351S and N421S;   (14) S254T, L306I, K326R and Q362L;   (15) H224Y, P230S, V323A, E333D, K338R and S364C;   (16) T335I, K414M and P445R;   (17) T335I and K414M;   (18) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A and L365P;   (19) H268N, V279M, A339T, N361D and S426P;   (20) C261Y, K290E, L306F, Q311R, E333G and Q438L;   (21) E283G, N315K, E333G, R344Q, L365P and S442T;   (22) Q347R, N361Y and K439R;   (23) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P and K447E; and   (24) E269G, Y278H, N325S and K370R,   wherein the numbering of the residues is that of the EU index as in Kabat.   
     
     
         38 . (canceled) 
     
     
         39 . The Fc polypeptide variant of  claim 35 , wherein the Fc ligand is an Fcγ receptor or C1q. 
     
     
         40 . The Fc polypeptide variant of  claim 39 , wherein the Fcγ receptor is selected from FcγRI, FcγRII or FcγRIII families. 
     
     
         41 - 46 . (canceled) 
     
     
         47 . The Fc polypeptide variant of  claim 35 , wherein the altered effector function is improved ADCC or CDC. 
     
     
         48 - 67 . (canceled) 
     
     
         68 . A method of increasing the percentage of Fc polypeptides comprising a mature core carbohydrate structure which lacks fucose present in a composition to at least 20%, said method comprising:
 a. introducing at least one mutation into a nucleic acid encoding the Fc polypeptide, wherein the mutation results in a substitution at amino acid residue 243; and   b. expressing the mutated nucleic acid in an animal cell to produce a glycosylated composition of Fc polypeptides,   
       wherein the numbering of the residues is that of the EU index as in Kabat. 
     
     
         69 - 70 . (canceled) 
     
     
         71 . The method of  claim 68 , wherein the mutation results in a substitution at position 243 selected from the group consisting of 243L, 243G, 243H and 243I. 
     
     
         72 - 73 . (canceled) 
     
     
         74 . The method of  claim 68 , wherein the percentage of Fc polypeptides comprising a mature core carbohydrate structure which has sialic acid present in the composition is increased to at least 10%. 
     
     
         75 - 76 . (canceled) 
     
     
         77 . The method of  claim 68 , wherein the percentage of Fc polypeptides comprising a mature core carbohydrate structure which has bisecting GlcNAc present in the composition is increased to at least 5%. 
     
     
         78 . A method of increasing the percentage of Fc polypeptides comprising a mature core carbohydrate structure which has sialic acid present in a composition to at least 10%, said method comprising:
 a. introducing at least one mutation into a nucleic acid encoding the Fc polypeptide, wherein the mutation results in a substitution at amino acid residue 243; and   b. expressing the mutated nucleic acid in an animal cell to produce a glycosylated composition of Fc polypeptides,   
       wherein the numbering of the residues is that of the EU index as in Kabat. 
     
     
         79 - 80 . (canceled) 
     
     
         81 . The method of  claim 78 , wherein the mutation results in a substitution at position 243 selected from the group consisting of 243L, 243G, 243H and 243I.